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Seventy-one non-pathogenic bacterial strains were surveyed for the presence of Type II restriction endonucleases in aerobic culture. Five strains were found to contain specific enzymes : Cfl I from Cellulomonas flavigena, Gal I from Gluconobacter albidus, Gce I from Gluconobacter cerinus, Hac I from Halococcus acetoinfaciens, and Mfl I from Microbacterium flavum. These enzymes cleaved respectively the sequences of 5'-CTGCA↓G-3', 5'-CCGC↓GG-3', 5'-CCGC↓GG-3', 5'-↓GATC-3', and 5'-Pu↓GATCPy-3' at the positions indicated. Hac I and Mfl I digested DNA from the Escherichia coli Dam^- strain but not from the Dam^+ strain, indicating that they react only with unmodified sequences. With NaCl or KCl, the optimal salt concentrations were 40-60 mM for Cfl I and 175-300 mM for Hac I; Gal I and Gce I did not require either salts. Activity was greatest at pH 7.1-7.5 for Cfl I and Hac I, and at pH 7.5-8.0 for Gal I and Gce I.